ATP sensitive potassium channels (KATP) in surface membrane (sKATP) and mitochondria (mKATP) of heart have both been implicated in the protective phenomenon called ischemic preconditioning (IPC). Proposed mechanisms for IPC are complex. The relative importance of mKATP and sKATP for IPC is controversial, in part because of heavy reliance on KATP pharmacology as well as the unknown molecular nature of mKATP. The sulfonylurea receptor subunit SUR2 regulates pharmacology and nucleotide interactions for sKATP, but the role of SUR2 in mKATP is unknown. In the last project periods we discovered novel SUR2 protein variants (Kd150, 68, 55, 28), one of which SUR2-55 was found uniquely in mitochondria. When we cloned and expressed SUR255 in COS-1 cells, it trafficked to mitochondria, but when we mutated a predicted mitochondrial targeting sequence, it trafficked to the membrane and when co-expressed with inward rectifier K channels KIR6 it caused a KATP current with decreased ATP sensitivity. A transgenic mouse model disrupting exons 12-16 (SUR2ex12-16) was constitutively protected from myocardial ischemia; it lacked long form SUR2 but retained SUR2-55 variants. We showed that the mitochondria from SUR2-ex12-16 mice had increased K+ flux and a protected mitochondrial phenotype of mild depolarization, resistance to Ca2+ overload, and signaling levels of reactive oxygen species (ROS). We created SUR2-55 over-expressing mice that also demonstrated constitutive myocardial protection and a protected mitochondrial phenotype. A transgenic mouse targeting exon 3 removed SUR proteins including SUR2-55 but died within 14 days after birth. Together these data strongly implicate SUR2 variants in general and SUR2-55 mitochondrial variants in particular as critically important for normal mitochondrial function and myocardial protection in transgenic mice models. But are SUR2 variants important for IPC in general? Our guiding and over-arching hypothesis is that a shift to SUR2-55 based mKATP is a key factor in the mechanism of IPC by increasing mitochondrial K flux, causing mild depolarization of mitochondrial membrane potential, thus triggering protective signaling cascades. This hypothesis will be addressed in three aims: 1) The mKATP aim is to discover the protein partners of SUR2-55 that form mKATP and determine the structure, function and pharmacology. 2) The transgenic mouse aim is to characterize a mouse that conditionally targets exon3 in heart, and test the hypothesis that the expected deleterious phenotype can be rescued by SUR2-55 alone. 3) The IPC aim is to determine the amount, locations, associations, and function of SUR2 variants, especially SUR2-55, in IPC. The molecular nature of mKATP is a key gap in knowledge, and its role in IPC is controversial. This proposal utilizes transgenic animals as a novel non-pharmacological approach to fill these gaps and address the controversy. The results may suggest more specific therapeutic targets in the SUR2 variants, and it will also provide important new information on mitochondrial structure and function in heart.